Kidney International, Vol. 66 (2004), pp.

633–640

Dopamine D2-like receptor agonist bromocriptine protects

against ischemia/reperfusion injury in rat kidney
VIHANG NARKAR,1,2 OKSANA KUNDUZOVA,2 TAHIR HUSSAIN, CLAUDIE CAMBON,
ANGELO PARINI, and MUSTAFA LOKHANDWALA
Heart and Kidney Institute College of Pharmacy, University of Houston, Houston, Texas; and INSERM U 388,
Pharm. Moleculaire et Physiopathologie Renale Institut Louis Bugnard, Toulouse, France

Dopamine D 2 -like receptor agonist bromocriptine protects
against ischemia/reperfusion injury in rat kidney.
Background. Dopamine, via activation of D 1 -like and D 2 -
like receptors, plays an important role in the regulation
of renal sodium excretion. Recently, we demonstrated that
dopamine D 2 -like receptor agonist (bromocriptine) stimu-
lates p44/42 mitogen-activated protein kinases (MAPKs) and
Na+ ,K+ ATPase (NKA) activity in proximal tubular epithe-
lial cells. Since both these parameters are compromised in is-
chemia/reperfusion (I/R) injury to the kidney, we investigated
whether bromocriptine protects against the injury.
Methods. In this study we used unilateral rat model of renal
I/R injury. The Sprague-Dawley rats were divided into vehi-
cle and bromocriptine groups. The vehicle and bromocriptine
group was treated with vehicle and bromocriptine (500 lg/kg
intravenously), respectively, 15 minutes before the induction of
unilateral ischemia followed by 24- or 48-hour reperfusion. At
the end of 24 or 48 hours the animals were sacrificed to collect
control and ischemic kidney cortices, in which necrosis, apop-
tosis, NKA activity, NKA a1 subunit expression, and p44/42
MAPK phosphorylation were measured.
Results. We found extensive necrosis, apoptosis, and de-
creased NKA activity (with no change in a1 subunit) in the is-
chemic kidney cortex compared to the nonischemic cortex from
the vehicle-treated rats as early as 24 hours post-reperfusion. In
contrast, I/R injury–induced necrotic, apoptotic, and decrease in
NKA activity were absent in the outer cortex of bromocriptine-
treated rats after 24 or 48 hours. Interestingly, we detected
significantly higher phosphorylation of p44/42 MAPKs in con-
trol and ischemic kidneys of bromocriptine-treated rats com-
pared to those of vehicle-treated rats.
1 Dr. Narkar’s present address is Integrative Biology and Pharmacology,
University of Texas Medical School-Houston, 6431 Fannin St., Houston,
TX 77030.
2 These authors contributed equally to this work.
Key words: bromocriptine, p44/42 MAPK, ischemia/reperfusion injury,
kidney.
Received for publication September 11, 2003
and in revised form January 2, 2004, and February 16, 2004
Accepted for publication March 1, 2003


C 2004 by the International Society of Nephrology
Conclusion. Therefore, bromocriptine, a D 2 -like receptor ag-
onist, may protect against I/R injury to proximal tubules of the
kidney, via p44/42 MAPK activation.
Ischemia/reperfusion (I/R) injury in kidney is one of
the common causes of acute renal failure. Renal I/R in-
jury is usually associated with decreases in renal blood
flow (RBF) and glomerular filtration rate (GFR), an in-
crease in fractional excretion of sodium, and increased
serum creatinine and blood urea nitrogen (BUN) levels
[1–5]. These functional defects in the ischemic kidney are
in part caused by necrosis and apoptosis of the tubular ep-
ithelial cells during I/R injury [6–8]. It has been suggested
that proximal tubules and medullary thick ascending limb
sustain maximum damage during I/R injury, with proxi-
mal tubules being most susceptible of the two [9–11].
Multiple cellular factors are involved in the post-I/R
injury events, which lead to acute renal failure [12–14].
Recently, several groups have begun to explore the role
of mitogen-activated protein kinases (MAPKs) in I/R in-
jury [4, 5, 10, 15–17]. MAPKs are a class of protein ki-
nases, which play an important role in the determination
of cell survival. While p44/42 MAPKs attenuate apopto-
sis, p38 and JNK promote apoptosis [18]. The differential
regulation of these three MAPKs is reported in I/R injury
to cardiac myocytes [16]. Further, Di Mari et al demon-
strated that differential regulation of p44/42, p38 and JNK
MAPKs determines the fate of the tubular epithelial cells
in renal I/R injury [10]. More specifically, selective acti-
vation of JNK, but not of p44/42 MAPK, leads to ex-
tensive apoptosis observed in the proximal tubules after
I/R injury [10, 17]. Therefore, drugs that activate p44/42
MAPKs and/or inactivate JNK may protect the kidney,
especially the proximal tubules, against I/R injury.
Dopamine plays an important role in the regulation
of renal function [19, 20]. Moreover, the in vivo and in
vitro pharmacologic effects of dopamine receptor ago-
nists in the kidney have been extensively studied [19, 20].
We recently showed that bromocriptine (D 2 -like receptor

633
634 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury
agonist) stimulates p44/42 MAPKs in the proximal
tubules of the kidney [21, 22], which is a potential target
to counter I/R injury. Since proximal tubules are maxi-
mally affected in the course of renal I/R injury [9–11], we
hypothesized that bromocriptine may protect against I/R
injury to the proximal tubules of the kidney.
The proposed hypothesis was tested using unilateral rat
model of renal I/R injury. In this model, one kidney serves
as a sham control and the other kidney is subjected to
45 minutes of ischemia followed by reperfusion. Using the
unilateral rat model, we previously showed that the kid-
ney subjected to I/R injury undergoes extensive necrosis
and apoptosis compared to sham operated kidney within
24 hours after reperfusion [17, 23]. Therefore, in studying
the reno-protective effects of bromocriptine, we evalu-
ated necrosis and apoptosis in rat kidney as biomarkers
of I/R injury. Moreover, in terms of sodium handling, I/R
injury induces the loss of major sodium transporters in
the kidney and leads to an increase in fractional sodium
excretion [2]. Interestingly, bromocriptine increases the
activity of at least one such transporter Na+ , K+ ,
ATPase (NKA) via p44/42 MAPK pathway in renal prox-
imal tubules [22]. On the basis of this observation, we ad-
ditionally measured the effect of bromocriptine on NKA
activity and protein expression in I/R injury.
The experiments were performed in Sprague-Dawley
rats treated with vehicle or bromocriptine before in-
duction of I/R injury. In both groups, the above three
parameters were measured in the outer renal cortical re-
gions (rich in proximal tubules) of sham control and I/R
injury kidney. Furthermore, p44/42 phosphorylation (ac-
tivation) was also measured in both groups to examine
whether its induction coincided with the protective ef-
fects of bromocriptine in I/R injury.
METHODS
All animal experiments were carried out according to
the principles of Laboratory Animal Care formulated by
the National Society for Medical Research and the Guide
for the Care and Use of Laboratory Animals (National
Institutes of Health publication 86–23, revised 1989, au-
thorization 00577, 1989, Paris, France). Male Sprague-
Dawley rats weighing 140 to 190 g (Harlan ZI Du
Malcourlet, France) were housed individually in standard
laboratory cages with ad libitum access to food and water.
Unilateral renal I/R injury in Sprague-Dawley rats
In this procedure, the rats were anesthetized with
sodium pentobarbital (60 mg/kg intraperitoneally). Next,
the jugular vein was cannulated with a polyethylene
catheter (PE-10) for the administration of the drug. Is-
chemia was induced by clamping the right renal artery
with nontraumatic vascular clip for 45 minutes. During
Unilateral
ischemia
15 min 45 min 24 hr or 48 hr
Group I
vehicle Reperfusion Sacrifice
Group II Histology
bromocriptine TUNEL
(500 µg/kg i.v.)
NKA activity
p44/42 MAPKs
Fig. 1. Schematic representation of induction of renal ischemic/
reperfusion (I/R) injury.
the operation, the animals were kept on a surgical ther-
mostatically controlled table at 38 ± 1◦ C under anesthe-
sia. After surgery the animals were returned to the cages,
where they had free access to food and water. The rats
were divided into two sets (24-hour and 48-hour sets),
with each set having two groups. In each set, one group
of animals (N = 6) was given bromocriptine (500 lg/kg
intravenously) 15 minutes prior to the renal artery occlu-
sion, whereas a second group (N = 6) received vehicle.
Experimental protocol is explained in Figure 1. The con-
tralateral nonischemic kidney serves as a control to the is-
chemic kidney. The kidneys from 24- or 48-hour set of rats
were removed after 24 hours or 48 hours of reperfusion,
respectively, and divided lengthways into two parts. One
part was suspended in ice-cold modified Krebs-Henseleit
buffer (KHB) solution [22] for biochemical assays and
the second was fixed in Dubose solution for histologic
examination.
Histologic examination
For histologic examination, Dubose-fixed and paraffin-
embedded kidney specimens (5 lm) were stained with
hematoxylin and eosin. Histologic changes were evalu-
ated by measurement of tissue necrosis graded on an A
to F scale in relation to the extent of kidney damage: A,
none; B, up to 10%; C, from 10% to 25%; D, from 25% to
50%; E, from 50% to 75%; and F, more than 75%. Tubu-
lar necrosis was assessed in the renal cortex at 24 hours
and 48 hours after I/R.
TUNEL assay
The sections of kidney embedded in paraffin were de-
paraffinized in toluene and a graded series of ethanol.
DNA fragmentation (apoptosis) was visualized in situ
on fixed tissues by the terminal transferase-mediated
deoxyuridine triphosphate (dUTP) nick-end labeling
(TUNEL) procedure, using the apoptosis detection
kit of Promega (Madison, WI, USA). Briefly, the de-
paraffinized sections were incubated in proteinase K
 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury 635

(20 lg/mL) solution to permeabilize the tissues, rinsed, Table 1. Histologic changes
and further fixed in paraformaldehyde (4%). The sections Vehicle + I/R Bromocriptine + I/R
Grading Tubular
were incubated with 1 lL of terminal deoxynucleotidyl scale necrosis % 24 hours 48 hours 24 hours 48 hours
transferase (25 U/lL) and fluorescein-12-dUTP in equili-
A 0 – – 4 3
bration buffer [25 mmol/L Tris-HCl, pH 6.6; 200 mmol/L B 0–10 – – 1 2
potassium cacodylate, pH 6.6, 2.5 mmol/L cobalt chlo- C 10–25 1 – – –
ride, 0.25 mg/mL bovine serum albumin (BSA), and 0.2 D 25–50 1 1 – –
mmol/L dithiothreitol (DTT)] for 1 hour at 37◦ C. Further, E
F
50–75
>75
2
1
2
2
–
–
–
–
the slides were rinsed four times in standard sodium cit-
I/R is ischemic/reperfusion injury. Histologic changes were evaluated by
rate (SSC) and phosphate-buffered saline (PBS) and im- measuring of tissue necrosis graded on a 0 to 5 scale in relation to the extent
mersed in 40 mL propidium iodide solution (1 lg/lL) for of kidney damage: A, none; B, up to 10%; C, from 10% to 25%; D, from
25% to 50%; E, from 50% to 75%; and F, more than 75%. The grading was
15 minutes. The positive control was treated by DNAse I performed in cortical regions of ischemic kidneys from vehicle-treated and
(1 lg/lL) before being processed with the TUNEL pro- bromocriptine-treated rats.
cedure. The TUNEL-positive cells were counted under
fluorescence microscope and data represented as percent
of cells that stained TUNEL positive. (0.05 mL), bromophenol blue (0.02 mL), corticular ho-
Preparation of cortical homogenate mogenate (0.1 mL), and water (0.03 mL). Further, the
loading samples were resolved by SDS-polyacrylamide
The kidneys isolated from the above groups were im- gel electrophoresis (PAGE) and transferred to polyvinyli-
mediately transferred to 5 mL ice-cold KHB solution dene fluoride (PVDF) membrane. The NKA a1 sub-
containing protease inhibitor cocktail. Outer cortex was units, phospho-p44/42 MAPKs, and total MAPKs on
separated from each kidney, to obtain the portion rich the PVDF membrane were detected using mono-
in proximal tubules. The isolated cortical slices were ho- clonal NKA a1-subunit antibody (Research Diagnos-
mogenized using Wheaton’s homogenizer at setting 7 tics, Flanders, NJ, USA), anti-phospho-p44/42 MAPKs
for 20 strokes, and the protein concentration of the ho- antibody (Promega), and anti-p42 MAPKs antibody
mogenate measured. The NKA activity, NKA a1-subunit (Santa Cruz Biotechonology, Santa Cruz, CA, USA),
expression, and p44/42 MAPKs phosphorylation were respectively. Where indicated, the Western blots were
measured in the cortical homogenate. subjected to densitometric analysis using Scion Image
NKA activity in cortical homogenate software. The MAPKs data are represented as the den-
sitometric ratio of phospho- to total-p42 MAPKs.
The NKA activity was measured in cortical ho-
mogenate (1 mg/mL) as previously described [28]. The
reaction mixture was carried out in 1.025 mL of as- Data analysis
say buffer [37.5 mmol/L immidazole buffer, 70 mmol/L Where applicable, data are presented as mean ± SEM
NaCl, 5 mmol/L KCl, 1 mmol/L Na ethylenediaminete- of number (N) of experiments. Statistical analysis was
traacetic acid (EDTA), 5 mmol/L MgCl 2 , 6 mmol/L done using unpaired Student t test or one-way analysis
NaN 3 , 75 mmol/L Tris-HCl] using 0.1 mL (0.1 mg pro- of variance (ANOVA) as explained in the figure legends.
tein) of cortical homogenate. The reaction was initi- The difference was considered statistically significant at
ated by adding 4 mmol/L adenosine triphosphate (ATP). P < 0.05.
Ouabain-insensitive ATPase activity was determined in
parallel using an assay buffer containing 150 mmol/L
RESULTS
Tris-HCl, 1 mmol/L ouabain instead of NaCl and KCl.
The reaction was carried out at 37◦ C for 15 minutes Necrosis in the cortical slices of
and terminated by adding 0.05 mL of ice-cold 50% vehicle- and bromocriptine-treated rats
trichloroacetic acid (TCA). The NKA activity was mea- The histologic alterations induced by unilateral renal
sured as the function of liberated inorganic phosphate ischemia were evaluated at 24 hours and 48 hours of
(Pi) in triplicates. The NKA activity was calculated as reperfusion. Extensive necrosis was observed in outer
the difference between the total and ouabain-insensitive cortex of ischemic compared to contralateral kidneys of
ATPase activity and was represented as Pi lmol/mg vehicle-treated rats after 24 hours of reperfusion (Fig. 2A
protein/min. and B, Table 1) and 48 hours of reperfusion (Table 1).
On the other hand, the histology of ischemic cortex from
NKA a1 subunits and p44/42 MAPKs in cortical bromocriptine (500 lg/kg intravenously)-treated rats was
homogenate by Western blotting similar to contralateral kidneys after 24 hours (Fig. 2C
Loading samples were prepared for western blotting and D, Table 1) and 48 hours (Table 1) of reperfusion.
containing sodium dodecyl sulfate (SDS)-Laemmli (4×) Bromocriptine did not produce any detectable histologic
636 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury

A B

Vehicle

Contralateral Ischemia

C D
Fig. 2. Histology in cortical region of kidney
slices of unilateral I/R rat model. Representa-
tive light micrographs (magnification, 40×) of
hematoxylin and eosin stained sections show-
ing morphologic features of cortical region 24
Bromocriptine hours post I/R injury. In vehicle-treated rats,
the ischemic cortical slices (B) showed ex-
tensive necrosis (indicated by arrows) com-
pared to the contralateral kidney (A). The
histomorphology of bromocriptine-treated is-
chemic kidneys (D) was normal and indistin-
Contralateral Ischemia guishable from contralateral control kidneys
(C). Similar results were obtained from 5 an-
Mag, 40× imals.

A B

Vehicle

Contralateral Ischemia

C D

Bromocriptine

Contralateral Ischemia
Fig. 3. Apoptosis in cortical region of kid-
E ney slices of unilateral ischemic/reperfusion
100 (I/R) injury rat model. Apoptosis was deter-
TUNEL-positive cells,

* # mined in 24 hours post-I/R cortical kidney

slices. Compared to the contralateral cortical
75 slices (A), in vehicle-treated rats, the ischemic
cortical slices (B) showed extensive TUNEL-
%

50 positive staining. (C) The contralateral cor-

tical slices of bromocriptine-treated rats are
represented. In bromocriptine-treated rats
25 low TUNEL-positive staining was detected in
the ischemic cortical slices (D). (E) Data rep-
0 resented as % TUNEL-positive cells (N = 3).
Co IR Co IR ∗ and # represent statistically significant dif-
Vehicle treated Bromocriptine ference between groups compared (P < 0.05)
[one-way analysis of variance (ANOVA); post
treated hoc, Tukey’s multiple comparision test].
 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury 637

A * B A

Na+, K+, ATPase,

Na+, K+, ATPase,

0.05

Pi µmol/mg/min
0.04
Pi µmol/mg/min
1 2 3 4
0.04
0.03 phospho-p44/42
0.03
0.02 0.02
0.01 0.01 Total-p42
0.00 0.00
Co IR Co IR Vehicle Bromocriptine
Vehicle treated Bromocriptine
treated B
1.5
C *

phospho-p42/total-p42
*
Co IR Co IR *
*

phosphorylation,
NKA α1 subunit 1.0

p42 MAPK
Veh Br

Fig. 4. Detection of NKA activity and a1 subunits in renal cortical

homogenates of unilateral ischemic/reperfusion (I/R) rat model. NKA
activity and a1 subunit levels were determined in cortical homogenates
obtained from the vehicle-treated and bromocriptine-treated rats 24
hours post-I/R. (A) In vehicle-treated rats, the NKA activity in is-
chemic cortical homogenate (IR) was significantly lower than in the
contralateral cortical homogenate (Co). (B) In bromocriptine-treated
rats, NKA activity was similar between contralateral and ischemic cor-
tical homogenates. Data are represented as inorganic phosphate (Pi)
released in lmol/mg/min. ∗ Represents significant difference between
Co and IR in vehicle-treated rats (P < 0.05) (unpaired Student t test).
(C) Representative Western blot of NKA a1 subunit expression in cor-
tical homogenates of vehicle-treated and bromocriptine-treated rats 24
hours post I/R. Similar results were obtained in N = 4 animals.
abnormalities in the contralateral kidney. This result in-
dicates that bromocriptine prevented cortical necrosis in
renal I/R injury after 24 and 48 hours post-reperfusion.
Apoptosis in the cortical slices of
vehicle- and bromocriptine-treated rats
Apoptosis was detected by TUNEL assay performed in
the cortical slices from the contralateral and ischemic kid-
neys. Extensive TUNEL-positive staining was observed
in the outer cortex of ischemic compared to contralateral
kidney of vehicle-treated rats (Fig. 3A and B). In con-
trast, low level of TUNEL-positive staining was detected
in contralateral and ischemic kidneys of bromocriptine-
treated rats (Fig. 3C and D). The percent of cells that
were TUNEL-positive in each group is represented in
Figure 3E. This result indicates that bromocriptine pre-
vented cortical apoptosis in renal I/R injury.
NKA activity in the cortical homogenates of
vehicle- and bromocriptine-treated rats
NKA activity was measured as described in the cortical
homogenates of the kidney. It was found that the NKA
activity in the cortical homogenate from the ischemic
kidney was decreased as compared to the contralateral
kidney in vehicle-treated rats (Fig. 4A). NKA activity
in the bromocriptine-treated rats was same in both the
contralateral and the ischemic kidney. Moreover, this ac-
tivity was similar to that of the contralateral kidney of
vehicle-treated rats (Fig. 4B). This result indicates that
0.5
0.0
1 2 3 4
Vehicle Bromocriptine
Fig. 5. Determination of p44/42 mitogen-activated protein kinases
(MAPKs) phosphorylation in renal cortical homogenates of unilateral
ischemic/reperfusion (I/R) rat model. (A) In vehicle-treated rats, low
phosphorylation was detected in contralateral (1) and ischemic (2) corti-
cal homogenate. On the other hand, bromocriptine-treated rats showed
extensive phosphorylation in contralateral (3) and ischemic (4) cortical
homogenate (upper panel). The total-p42 MAPKs was similar in all the
groups compared (1, 2, 3, and 4) (lower panel). The above are represen-
tative blots. Similar results were obtained from N = 4 sets of animals.
(B) Data represented as average (mean ± SEM) ratio of phospho-p42
to total p42 MAPKs from N = 4 animals. (P < 0.05) [one-way analysis
of variance (ANOVA); post hoc, Newman-Keuls multiple comparison
test).
bromocriptine prevents the loss of NKA activity in the
cortex of the ischemic kidney.
To test the possibility that change in the NKA ac-
tivity is due to modified protein levels, we measured
the expression of catalytic NKA a1 subunit in the
above homogenates. We did not detect any difference
in the expression of NKA a1 subunits in the contralat-
eral or ischemic cortical homogenates from vehicle and
bromocriptine-treated animals (Fig 4C). Therefore, the
changes in NKA activity are independent of the level of
NKA a1 subunits in the cortex.
The p44/42 MAPKs activity in the
vehicle- and bromocriptine-treated rats
The p44/42 MAPKs activity was measured as the func-
tion of p44/42 MAPKs phosphorylation in the cortical ho-
mogenates of the kidney. In the vehicle-treated rats, there
was low detectable phosphorylation of p44/42 MAPKs in
both the control and ischemic cortices (Fig. 5A, upper
panel). The total p42 MAPKs in all the homogenates
638 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury
was similar (Fig. 5A, lower panel). In bromocriptine-
treated rats the phosphorylation of p44/42 MAPKs was
similar in both the contralateral and ischemic kidney
(Fig. 5A, upper panel). Moreover, the phosphoryla-
tion of p44/42 MAPKs in the bromocriptine-treated rats
was significantly higher than the phosphorylation in the
vehicle-treated rats. This was not simply due to more
p44/42 MAPKs protein, as the total protein loaded was
same in vehicle-treated and bromocriptine-treated rats
(Fig. 5A, lower panel). Thus, bromocriptine increases
phospho-p44/42 MAPKs in both contralateral and
ischemic cortices of bromocriptine-treated rats as com-
pared to vehicle-treated rats. The above data are repre-
sented as densitometric ratio of phospho-p42 to total-p42
MAPKs (Fig. 5B).
DISCUSSION
This study was designed to explore the potential of syn-
thetic dopamine D 2 -like receptor agonist, bromocriptine,
to protect against experimental I/R injury to the renal
proximal tubules. We found that I/R injury caused necro-
sis, apoptosis, and loss of NKA activity in the outer cortex
of the kidney, which is rich in proximal tubules. Inter-
estingly, pretreatment of the animals with bromocriptine
prevented necrosis, apoptosis, and loss of NKA activity
associated with I/R injury in proximal tubular regions of
the kidney. This protective effect may be due to the ac-
tivation of p44/42 MAPKs in the bromocriptine-treated
kidney.
I/R injury is known to induce differential activation
of various MAPKs in different segments of the kidney
nephron [10]. Thus, selective activation of JNK leads to
apoptosis of tubular cells, whereas activation of p44/42
MAPKs renders the cells viable [10]. It was suggested
that I/R injury leads to activation of JNK and not p44/42
MAPKs in proximal tubules, making them more sus-
ceptible to apoptosis [10, 17]. In another study, a brief
ischemic preconditioning [4] to kidney or urethral ob-
struction [5] prior to a second ischemia protected the kid-
ney morphologically, as well as functionally against I/R
injury. In these studies it was found that either pretreat-
ment primes the kidney such that, during the second I/R
injury there is decreased p38/JNK activation and/or in-
creased p44/42 MAPKs stimulation. Alternatively, phar-
macologic interventions which protect against renal I/R
injury also increase p44/42 MAPKs in the kidney [10,
17]. Since bromocriptine activates p44/42 MAPKs in re-
nal proximal tubular cells [21, 22], which are known to be
maximally susceptible to I/R injury, it was logical to study
the effects of bromocriptine in this pathophysiologic
situation.
We measured the effect of bromocriptine pretreatment
on I/R injury–induced necrosis, apoptosis, and down-
regulation of NKA activity in the proximal tubules of
the kidney. We found that unilateral I/R injury to kid-
ney caused extensive necrosis by 24 hours in the cortex
(rich in proximal tubules) of ischemic kidney as com-
pared to the control kidney. This observation was in
concert with previous reports [8, 17, 23]. When the ani-
mals were pretreated with bromocriptine, the kidney did
not undergo necrosis, indicating the protective effect of
bromocriptine.
In addition to necrosis, unilateral I/R injury caused
apoptosis in the cortex of the ischemic kidney as com-
pared to the control kidney in the vehicle-treated rats.
Pretreatment of the rats with bromocriptine prevented
I/R injury–induced apoptosis in the kidney. This pro-
tective effect of bromocriptine can be attributed to ac-
tivation of p44/42 MAPKs. As mentioned above, JNK
is predominantly activated in the cortex during I/R in-
jury, which predisposes the proximal tubules to apoptotic
death [10]. By activating p44/42 MAPKs, which are anti-
apoptotic, bromocriptine might cancel out or balance the
effect of JNK.
In concert with some previous reports [24, 25], we
found that the NKA activity in the ischemic cortex was
decreased as compared to the contralateral nonischemic
cortex. However, our results contradict alternative stud-
ies which report no change or actual increase in the NKA
activity in the kidney [26, 27]. This discrepancy may be
associated with the differences in the models of I/R injury
used or due to the use of whole kidney lysate versus cor-
tical lysates for measuring NKA activity. Nevertheless,
pretreatment of the animals with bromocriptine in our
experiments prevented the loss of NKA activity in I/R in-
jury. Unlike the NKA activity, the expression of catalytic
NKA a1 subunit remained constant in all the treatment
groups. The consistent expression of NKA a1 subunit
suggests that bromocriptine restores NKA activity with-
out changing the protein expression. Interestingly, earlier
studies done in our laboratory show that bromocriptine
stimulates NKA activity via p44/42 MAPKs-dependent
recruitment of NKA a1 subunits to the proximal tubular
membrane in the kidney [21, 22, 28, 29]. It is possible that
bromocriptine may prevent the loss of NKA activity in
renal I/R injury by similar mechanism. Moreover, mainte-
nance of NKA activity in the proximal tubular membrane
may partially prevent the increase in fractional excretion
of sodium observed during I/R injury [2].
As previously reported, we found that there was lower
phospho (active)-p44/42 MAPKs in ischemic cortex of
vehicle-treated animals [10]. On the other hand, ro-
bust increase in phospho-p44/42 MAPKs was observed
in both the ischemic and the control non-ischemic kid-
neys of bromocriptine-treated animals. Further, acti-
vation of p44/42 MAPKs in the bromocriptine-treated
animals were concomitant with the protection against
I/R injury (at the level of necrosis, apoptosis, and NKA
activity) in the same animals. In terms of proapoptotic
 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury
MAPKs, several drugs protect against I/R injury by de-
creasing JNK activity in addition to increasing that of
p44/42 MAPK [10, 17, 30–33]. However, in experiments
done in our lab, bromocriptine did not decrease the el-
evated JNK activity in the ischemic cortex (data not
shown). Therefore, bromocriptine may protect against re-
nal I/R injury by increasing antiapoptotic p44/42 MAPKs
activity, without additionally modifying proapoptotic
JNK activity.
It is noteworthy that certain studies suggest the in-
creased renal phospho-p44/42 MAPKs to be actually
responsible for I/R injury [31, 34]. Moreover, in the
same studies reno-protective agents such as nitric oxide
donors protect against renal I/R injury and simultane-
ously decrease phospho-p44/42 MAPKs in the ischemic
kidneys [31, 34]. At this point it is hard to explain the
discrepancy between these reports and our observations.
However, there are several differences between our ex-
periments and ones performed in these studies at levels
such as the model of I/R injury and time of phospho-
p44/42 MAPK measurement, which can account for
different observations. Moreover, as mentioned above,
we detected increased phospho-p44/42 MAPKs in the
control kidney of bromocriptine-treated animals, which
showed no signs of necrosis or apoptosis. Therefore, it
is more likely that p44/42 MAPKs are protective rather
than causative in renal I/R injury.
The activation of p44/42 MAPKs may not be the sole
mechanism for the protective effects of bromocriptine.
It is interesting to note that the protective effects of
bromocriptine and other dopamine D 2 -like receptor ag-
onist against cerebral I/R injury have been previously
reported [35, 36, 37]. Multiple mechanisms such as (1)
inhibition of dopamine release [38], (2) activation of su-
peroxide dismutase/free radical scavenging [39, 40, 41,
42, 43], and (3) activation of antiapoptotic factors [35]
are responsible for neuroprotective effects of dopamine
D 2 -like receptor agonists. It is likely that similar mecha-
nisms, in addition to the one we have described, may also
play a role in the protection of proximal tubules during
I/R injury.
CONCLUSION
Our results show that bromocriptine, a dopamine D 2 -
like receptor agonist, protects against renal I/R injury.
Further, this protective effect may primarily involve ac-
tivation of p44/42 MAPK. Additional studies are neces-
sary to explore the potential of bromocriptine in treating
post-I/R acute renal failure.
ACKNOWLEDGMENTS
This study was supported in part by NIH grant AG-15031 and by the
Foundation pour la Recherché Médical Française.
639
Reprint requests to Dr. Mustafa F. Lokhandwala, Heart and Kidney
Institute, College of Pharmacy, University of Houston, Houston, TX
77204–5041.
E-mail: Mlokhandwala@uh.edu
REFERENCES
1. CHIAO H, KOHDA Y, MCLEROY P, et al: Alpha-melanocyte-
stimulating hormone protects against renal injury after ischemia
in mice and rats. J Clin Invest 99:1165–1172, 1997
2. KWON TH, FROKIAER J, HAN JS, et al: Decreased abundance of major
Na+ transporters in kidneys of rats with ischemia-induced acute
renal failure. Am J Physiol Renal Physiol 278:F925–F939, 2000
3. MASHIACH E, SELA S, WINAVER J, et al: Renal ischemia-reperfusion
injury: Contribution of nitric oxide and renal blood flow. Nephron
80:458–467, 1998
4. PARK KM, CHEN A, BONVENTRE JV: Prevention of kidney
ischemia/reperfusion-induced functional injury and JNK, p38 and
MAPKs kinase activation by remote ischemic pretreatment. J Biol
Chem 276:11870–11876, 2001
5. PARK KM, KRAMERS C, VAYSSIER-TAUSSAT M, et al: Prevention of kid-
ney ischemia/reperfusion-induced functional injury, MAPKs and
MAPKs kinase activation, and inflammation by remote transient
urethral obstruction. J Biol Chem 277:2040–2049, 2002
6. BONEGIO R, LIEBERTHAL W: Role of apoptosis in the pathogenesis of
acute renal failure. Curr Opin Nephrol Hypertens 11:301–308, 2002
7. GOBE G, WILLGOSS D, HOGG N, et al: Cell survival or death in re-
nal tubular epithelium after ischemia-reperfusion injury. Kidney Int
56:1299–1304, 1999
8. PADANILAM BJ: Cell death induced by acute renal injury: A perspec-
tive on the contributions of apoptosis and necrosis. Am J Physiol
Renal Physiol 284:F608–F627, 2003
9. BREZIS M, ROSEN S, SILVA P: Renal ischemia: A new perspective.
Kidney Int 26(4):375–383, 1984
10. Di MARI JF, DAVIS R, SAFIRSTEIN RL: MAPKs activation determines
renal epithelial cell survival during oxidative injury. Am J Physiol
277:F195–F203, 1999
11. VENKATACHALAM MA, BERNARD DB, DONOHOE JF, et al: Ischemic
damage and repair in the rat proximal tubule: Differences among
the S1, S2, and S3 segments. Kidney 14(1):31–49, 1978
12. BRADY HR, BRENNER BM, LIEBERTHAL WL: Acute renal failure, in
The Kidney, 1986, pp 1200–1257, 1986
13. BONVENTRE JV: Mediators of ischemic renal injury. Ann Rev Med
39:531–544, 1988
14. RACUSEN LC: The morphological basis of acute renal failure, in
Acute Renal Failure 2001, pp 1–12
15. ABE J, BAINES CP, BERK BC: Role of mitogen-activated protein
kinases in ischemia and reperfusion injury. Circ Res 86:607–609,
2000
16. YUE TL, WANG C, GU JL, et al: Inhibition of extracellular signal-
regulated kinase enhances ischemia-reoxygenation-induced apop-
tosis in cultured cardiac myocytes and exaggerates reperfusion in-
jury in isolated perfused heart. Circ Res 86:692–699, 2000
17. KUNDUZOVA OR, BIANCHI P, PIZZINAT N, et al: Regulation of
JNK/ERK activation, cell apoptosis, and tissue regeneration by
monoamine oxidases after renal ischemia-reperfusion. FASEB J
16:1129–1131, 2002
18. XIA Z, DICKENS M, RAINGEAUD J, et al: Opposing effects of ERK and
JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331, 1995
19. HUSSAIN T, LOKHANDWALA MF: Renal dopamine receptors and hy-
pertension. Exp Biol Med 228:134–142, 2003
20. JOSE PA, EISNER GM, FELDER RA: Dopamine receptors in health
and hypertension. Pharmacol Ther 80:149–182, 1998
21. NARKAR VA, HUSSAIN T, PEDEMONTE C, et al: Dopamine D 2 receptor
activation causes mitogenesis via p44/42 mitogen-activated protein
kinase in opossum kidney cells. J Am Soc Nephrol 12:1844–1852,
2001
22. NARKAR VA, HUSSAIN T, LOKHANDWALA MF: Role of tyrosine kinase
and p44/42 MAPKs in D 2 -like receptor-mediated stimulation of
Na+ , K+ , ATPase in kidney. Am J Physiol: Renal Physiol 282:F697–
F702, 2002
23. KUNDUZOVA OR, BIANCHI P, PIZZINAT N, et al: Regulation of
JNK/ERK activation, cell apoptosis, and tissue regeneration by
640 Narkar et al: Bromocriptine and renal ischemia/reperfusion injury
monoamine oxidases after renal ischemia-reperfusion. FASEB J
16:1129–1131, 2002
24. KAJIWARA I, KAWAMURA K, HIRATSUKA Y, TAKEBAYASHI S: The
influence of oxygen free radical scavengers on the reduction
of membrane-bond Na+ -K+ -ATPase activity induced by is-
chemia/reperfusion injury in canine kidney. Nephron 72:637–643,
1996
25. AYDIN S, ARICIOGLU A, TURKOZKAN N, et al: Na+ ,K+ ,ATPase activity
of rabbit kidney cortex membranes in ischemia and reperfusion.
Acta Biochem Pol 40:545–547, 1993
26. FEKETE A, VANNAY A, VER A, et al: Gender differences in the alter-
ations of Na+/K+-ATPase following ischemia/reperfusion injury in
the rat kidney. J Physiol (Lond) 2003
27. WANG Z, RABB H, HAQ M: A possible molecular basis of natriuresis
during ischemic-reperfusion injury in the kidney. J Am Soc Nephrol
9(4):605–613, 1998
28. HUSSAIN T, ABDUL-WAHAB R, LOKHANDWALA MF: Bromocriptine
stimulates Na+ ,K+ ,ATPase in renal proximal tubules via the cAMP
pathway. Eur J Pharmacol 321:259–263, 1997
29. NARKAR VA, HUSSAIN T, LOKHANDWALA MF: Activation of D 2 -
like receptors causes recruitment of tyrosine phosphorylated
Na+ ,K+ ,ATPase a1-subunits in kidney. Am J Physiol Renal Physiol
283:F1290–F1295, 2002
30. DIMARI J, MEGYESI J, UDVARHELYI N, et al: N-acetyl cysteine ame-
liorates ischemic renal failure. Am J Physiol 272:F292–F298, 1997
31. MEHTA A, SEKHON CP, GIRI S, et al: Attenuation of is-
chemia/reperfusion induced MAP kinases by N-acetyl cysteine,
sodium nitroprusside and phosphoramidon. Mol Cell Biochem
240:19–29, 2002
32. YANG CW, LI C, JUNG JY, et al: Preconditioning with erythropoi-
etin protects against subsequent ischemia-reperfusion injury in rat
kidney. FASEB J 17:1754–1755, 2003
33. YANG CW, AHN HJ, JUNG JY, et al: Preconditioning with cy-
closporine A or FK506 differentially regulates mitogen-activated
protein kinase expression in rat kidneys with ischemia/reperfusion
injury. Transplantation 75:20–24, 2003
34. MARTINEZ-MIER G, TOLEDO-PEREYRA LH, MCDUFFIE JE, et al:
Exogenous nitric oxide downregulates MIP-2 and MIP-1alpha
chemokines and MAPK p44/42 after ischemia and reperfusion of
the rat kidney. J Invest Surg 15:287–296, 2002
35. KIHARA T, SHIMOHAMA S, SAWADA H, et al: Protective effect of
dopamine D 2 agonists in cortical neurons via the phosphatidyli-
nositol 3 kinase cascade. J Neurosci Res 70:274–282, 2002
36. LIU XH, KATO H, CHEN T, et al: Bromocriptine protects against
delayed neuronal death of hippocampal neurons following cerebral
ischemia in the gerbil. J Neurol Sci 29:9–14, 1995
37. O’NEILL MJ, HICKS CA, WARD MA: Dopamine D 2 receptor agonists
protect against ischemia-induced hippocampal neurodegeneration
in global cerebral ischemia. Eur J Pharmacol 352:37–46, 1998
38. TISSARI AH, LILLGÄLS MS: Reduction of dopamine synthesis inhibi-
tion by dopamine auto-receptor activation in striatal synaptosomes
with in vivo reserpine administration. J Neurochem 61:231–238,
1993
39. GLOVER V, CLOW A, SANDLER M: Effects of dopaminergic drugs on
superoxide dismutase: Implications for senescence. J Neural Transm
40 (Suppl):37–45, 1993
40. TAGAYA M, MATSUMOTO M, KITAGAWA K: Recombinant human su-
peroxide dismutase can attenuate ischemic neuronal damage in ger-
bils. Life Sci 51:253–259, 1992
41. WENGENACK TM, CURRAN GL, PODUSLO JF: Postischemic, systemic
administration of polyamine-modified superoxide dismutase re-
duces hippocampal CA1 neurodegeneration in rat global cerebral
ischemia. Brain Res 754:46–54, 1997
42. YOSHIKAWA T: Free radicals and their scavengers in Parkinson’s dis-
ease. Eur Neurol 33:60–68, 1993
43. YOSHIKAWA T, MINAMIYAMA Y, NAITO Y, et al: The antioxidant prop-
erties of dopamine agonist bromocriptine. J Neurochem 62:1034–
1038, 1994